HIF1A protein expression is correlated with clinical features in gastric cancer: an updated systematic review and meta-analysis

To elucidate the correlation of HIF1A with clinicopathologic characteristics in patients with gastric cancer (GC), we conducted a systematic review and meta-analysis. We searched PubMed, Embase and Web of Science for studies on GC and HIF1A, covering studies published until January 31st, 2022. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) for clinical characteristics based on high and low HIF1A protein levels. We used random-effects and fixed-effects meta-analysis methods to determine mean effect sizes of ORs and evaluated publication heterogeneity with τ2, I2, and Q values. Additionally, we generated funnel plots to inspect publication bias. Our meta-analysis included 20 publications with 3416 GC patients to estimate the association between high or low HIF1A expression and clinical characteristics. Positive HIF1A expression was significantly associated with T stage progression (OR: 2.46; 95% CI 1.81–3.36; P < 0.01), TNM stage progression (OR: 2.50; 95% CI 1.61–3.87; P < 0.01), lymph node metastasis (OR: 2.06; 95% CI 1.44–2.94; P < 0.01), undifferentiated status (OR: 1.83; 95% CI 1.45–2.32; P < 0.01), M stage progression (OR: 2.34; 95% CI 1.46–3.77; P < 0.01), Borrmann stage progression (OR: 1.48; 95% CI 1.02–2.15; P = 0.04), larger tumor size (OR: 1.27; 95% CI 1.06–1.52; P < 0.01), vascular invasion (OR: 1.94; 95% CI 1.38–2.72; P < 0.01), and higher vascular endothelial growth factor (VEGF) protein expression (OR: 2.61; 95% CI 1.79–3.80; P < 0.01) in our meta-analysis. GC Patients highly expressing HIF1A protein might be prone to tumor progression, poorly differentiated GC cell types, and a high VEGF expression.


Selection criteria
The following criteria were considered to select the publications: Inclusion criteria: 1. Case-control design studies in GC 2. GC studies on HIF1A protein levels using immunohistochemistry (IHC) staining 3. Studies having appropriate data for obtaining odds ratios (ORs) and their 95% confidence intervals (CIs).

Assessment of quality
The evidence of quality was assessed according to Robinson et al. 31 [Supplementary Table S1].

Effect measures and assessment of heterogeneity
For the meta-analysis, the R package "meta" 32 generated forest plots depicting ORs and 95% CIs from the selected studies.Random-effects or fixed-effects models (equivalently, common-effects model) assessed the pooled effect sizes of the ORs.The pooled effect size refers to the combined effect size estimates of the studies and is a crucial tool in determining the clinical association of high or low HIF1A protein levels in GC.Heterogeneity was calculated using the R library "meta": between-study variance τ 2 and Higgins' I 2 , including Cochran's Q-tests 32 .The fixed-effects models estimated pooled ORs when P ≥ 0.05 or I 2 ≤ 50%, indicating the absence of heterogeneity 29 .Otherwise, the random-effects models generated pooled ORs 29,33 .The forest plots depicted clinical outcomes of GC patients who had high vs. low HIF1A protein expression.
Overexpression of HIF1A is crucial in cancer cell migration and proliferation 6 .However, the impact of HIF1A protein expression on clinical characteristics related to cancer cell migration and proliferation has not been thoroughly investigated.The proliferation and migration of cancer cells promote advanced cancer stages and cytological features.Therefore, we examined the clinical parameters, including TNM stage advancement and cancer cell differentiation status.We also inspected other clinical features (i.e., gender, age, T stage progression, N stage progression [lymph node metastasis], M stage progression, Borrmann types, tumor sizes, VEGF status, vascular invasion, Lauren classification, and tumor sites).

Publication bias
Sensitivity analysis measured the effects of individual publications on the overall results by omitting one publication at a time.Subsequently, we examined the publication bias (standard error of OR vs. OR) using funnel plots and Egger's tests.The absence of skewness and asymmetry in funnel plots often implies the absence of publication bias.

Meta-regression
We employed a mixed-effects meta-regression model utilizing the R package "metafor" (version 4.4.0) 34to assess the impacts of covariates, age, and sex.Median ages and male-to-female ratios across individual studies were utilized as surrogate measures for age and sex, respectively.Meta-regression analysis was conducted for a given outcome when a covariate was present in a minimum of ten individual studies.We derived P-values to assess heterogeneity and to test the null hypothesis of no linear relationship between covariates and effect sizes in the meta-regression.Additionally, we presented bubble plots illustrating the associations of outcomes (i.e., ORs) with covariates.

Literature search and included studies
PubMed, Embase and Web of Science searches reported 646, 133 and 495 studies, respectively.We discovered 538 publications after deleting duplicates and thoroughly inspecting the titles and abstracts.Then, 518 studies were discarded because of a lack of data and an unclear number of patients according to the selection criteria.Finally, 20 papers were chosen for the meta-analysis (Fig. 1).
There was a total of 3416 patients in the 20 articles, including 1784 HIF1A-positive and 1632 HIF1A-negative individuals with GC.IHC was used to evaluate HIF1A protein expression in patients with GC in these 20 studies (Table 1).The clinical features included gender, age, T stage progression, N stage progression, M stage progression, Borrmann types, tumor sizes, VEGF status, vascular invasion, Lauren classification, and tumor sites.
Table 1.Included publications for HIF1A immunohistochemistry (IHC) meta-analysis.The 20 publications included to analyze HIF1A protein expression in this meta-analysis.In the studies, protein level was measured using immunohistochemistry (IHC).For each study, the HIF1A antibodies, IHC scoring schemes, and clinicopathological features were described.Study names for the meta-analysis are indicated in the first column.The other clinical features, including gender, age, tumor sites, and Lauren classification were not significantly associated with HIF1A expression in GC patients.
The Q-test P values for TNM stage progression, T stage progression, M stage progression, N stage progression, vascular invasion, VEGF positivity, Borrmann stage progression, differentiation status, and tumor size were < 0.01, < 0.01, 0.12, < 0.01, 0.75, 0.25, 0.38, < 0.01, and 0.41, respectively (Fig. 2).When the P value was < 0.05, the rejection of the null hypotheses of the same effect sizes in all studies indicated the effect sizes varied across studies.
Figure 2. Meta-analysis on clinical characteristics.Events indicate high HIF1A expressing patients with GC (equivalently, HIF1A-positive patients with GC).Events suggest that patients with GC express high HIF1A protein levels.The first through sixth column lists: the study names, experimental group, control group, forest plots, odds ratios (ORs) of patients in the experimental group with high levels of HIF1A protein compared with those of control group, with 95% CI, and weight.One pooled effect size, OR, for high vs. low HIF1A levels in patients with GC is indicated with a clinicopathological feature.Between-study variance τ 2 , Higgins' I 2 , and Cochran's Q-tests were used to quantify heterogeneity.(A) TNM stages III-IV as experimental group vs. I-II as control group.In a group, events (i.e., high HIF1A protein-expressing GC patients) were obtained from a given study.The overall pooled effect estimate suggests that the OR of high or low HIF1A protein level between the two groups was > 1. High HIF1A-expressing patients are more in the experimental group (i.e., stages III-IV) than that in the control group (I-II).Positive HIF1A expression is associated with advanced TNM stages.

Robustness and publication bias
The ORs were consistent when we performed sensitivity analysis of statistically significant clinicopathological characteristics (Fig. 3).Therefore, the sensitivity analysis for significant clinicopathological characteristics confirmed the robustness of the results.
The visual assessment of the funnel plots revealed no asymmetry, suggesting the possibility of publication bias (Fig. 4).Egger's tests indicated no statistical significance as well.Overall, the funnel plots and Egger's tests revealed no evidence of publication bias.

Meta-regression
Age exhibited a significant association with subgroups categorized by TMN stage progression, T stage progression, N stage progression, and differentiation statuses (Supplementary Fig. S1a-d), suggesting age as a source of heterogeneity.Conversely, while sex did not show a significant association with subgroups divided by T stage progression, it displayed a significant association with subgroups categorized by TMN stage progression, N stage progression, and differentiation statuses (Supplementary Fig. S1e-h), indicating sex as a source of heterogeneity in subgroups categorized by TMN stage progression, N stage progression, and differentiation statuses.

Discussion
The purpose of our study was to determine whether GC patients with high protein levels of HIF1A had more severe clinical features compared with those who did not.We found that the T stage progression, TNM stage progression, lymph node metastasis, differentiated status, M stage progression, Borrmann stage progression, tumor size, vascular invasion, and VEGF protein expression were significantly associated with GC patients with high levels of the HIF1A protein.
Our meta-analysis inspected 20 publications compared with nine studies evaluated in a previous meta-analysis of HIF1A protein expression in GC 23 , enhancing the knowledge of the correlation between HIF1A protein expression and clinical characteristics.Also, this meta-analysis revealed broader correlations between the protein expression and clinical characteristics when compared to the previous meta-analysis 23 .
HIF1A is a functionally important mediator in GC 6 .Activated HIF1A recruits M2-type tumor-associated macrophages (TAMs), aiding chemoresistance in GC 35 .It is also associated with resistance to anoikis-cell death due to the detachment from the extracellular matrix-in GC 36 .Inhibition of HIF1A in GC induced anoikis via integrin-5 36 .
HIF1A is also a mediator of EMT 6 which promotes tumor progression in the advanced stages 37 .In this meta-analysis, the association between cancer stage progression and positive HIF1A protein expression may support HIF1A-related EMT.EMT affects cellular morphology changes mostly by inhibiting genes involved in differentiation 37 .This aligns with our findings linking HIF1A protein expression to poorly differentiated GC. www.nature.com/scientificreports/Additionally, EMT contributes to the progression of the Lauren diffuse subtype in GC 38 .Considering the involvement of HIF1A in EMT 37 , the association between the diffuse subtype and positive HIF1A expression could be predicted, but the association was not statistically significant in our meta-analysis.A previous www.nature.com/scientificreports/meta-analysis comprising nine studies on HIF1A and clinicopathological features in GC 10 reported that positive HIF1A protein level was associated with TNM stage progression, differentiated GC cell status, T stage progression, vascular invasion, and lymph node metastasis.These results were consistent with our meta-analysis results.New findings of our meta-analysis indicated the statistical associations between positive HIF1A expression and other clinicopathological features, including Borrmann stage progression, positive VEGF protein expression, and tumor sizes.
Based on the functional roles of HIF1A and the results of our meta-analysis, HIF1A signaling is essential for GC progression, and the protein is a potential biomarker.Meta-analyses can provide information on HIF1A protein expression for patient classification.HIF1A is associated with PD-L1, a target of immune checkpoint inhibitors in cancer 39 .In the hypoxic tumor microenvironment, HIF1A binds to the PD-L1 promoter region, upregulating PD-L1 in myeloid-derived suppressor cells and tumor cells 40 .
We believe that this systematic review and meta-analysis provide a complete picture of the clinical association of HIF1A protein expression with GC.Also, the 3416 patients from the 20 studies offered statistical support for a reliable meta-analysis.
However, this study has some limitations.As we used published papers in our meta-analysis, publication bias is unavoidable, which means statistical heterogeneity is inescapable 41 .Publication bias may be due to different patient sources and IHC scoring schemes for measuring protein expression in the selected papers.Additionally, the HIF1A antibodies used to stain the protein varied (Table 1) and might contribute to publication bias.

Conclusions
According to our research, high levels of HIF1A protein in GC are associated with T stage progression, TNM stage progression, lymph node metastasis, differentiated status, M stage progression, Borrmann stage progression, tumor size, vascular invasion, and positive VEGF protein expression, providing potential biological indicators for the diagnosis and prognosis of patients with GC.Our findings suggest that prospective, large-scale cohort studies are needed to verify HIF1A protein level as a biomarker candidate for GC development.
Figure2.Meta-analysis on clinical characteristics.Events indicate high HIF1A expressing patients with GC (equivalently, HIF1A-positive patients with GC).Events suggest that patients with GC express high HIF1A protein levels.The first through sixth column lists: the study names, experimental group, control group, forest plots, odds ratios (ORs) of patients in the experimental group with high levels of HIF1A protein compared with those of control group, with 95% CI, and weight.One pooled effect size, OR, for high vs. low HIF1A levels in patients with GC is indicated with a clinicopathological feature.Between-study variance τ 2 , Higgins' I 2 , and Cochran's Q-tests were used to quantify heterogeneity.(A) TNM stages III-IV as experimental group vs. I-II as control group.In a group, events (i.e., high HIF1A protein-expressing GC patients) were obtained from a given study.The overall pooled effect estimate suggests that the OR of high or low HIF1A protein level between the two groups was > 1. High HIF1A-expressing patients are more in the experimental group (i.e., stages III-IV) than that in the control group (I-II).Positive HIF1A expression is associated with advanced TNM stages.(B) T stages T3-T4 vs. T1-T2.(C) M stages M1 vs. M0.(D) N stages N1-N3 vs. N0.(E) Positive vs. negative vascular invasion.(F) High (positive) vs. low (negative) VEGF expression.(G) Borrmann types 4-5 vs. 1-3.(H) Undifferentiated vs. differentiated differentiation statuses.(I) Tumor sizes ≥ 5 cm vs. < 5 cm.